The present invention relates to purification of organic compounds, and more particularly to the purification of 1xcex1-hydroxyvitamin D2 (1xcex1-OH-D2) by preparing it in crystalline form.
Purification of organic compounds, especially those designated for pharmaceutical use, is of considerable importance for chemists synthesizing such compounds. Preparation of the compound usually requires many synthetic steps and, therefore, the final product can be contaminated not only with side-products derived from the last synthetic step of the procedure but also with compounds that were formed in previous steps. Even chromatographic purification, which is a very efficient but relatively time-consuming process, does not usually provide compounds which are sufficiently pure to be used as drugs.
Depending on the method used to synthesize 1xcex1-hydroxyvitamin D compounds, different minor undesirable compounds can accompany the final product. Thus, for example, if direct C-1 hydroxylation of 5,6-trans geometric isomer of vitamin D is performed, followed by SeO2/NMO oxidation and photochemical irradiation [see Andrews et al., J. Org. Chem. 51, 1635 (1986); Calverley et al., Tetrahedron 43, 4609 (1987); Choudry et al., J. Org. Chem. 58, 1496 (1993)], the final 1xcex1-hydroxyvitamin D product can be contaminated with 1xcex1-hydroxy- as well as 5,6-trans isomers. If the method consists of C-1 allylic oxidation of the 4-phenyl-1,2,4-triazoline-3,5-dione adduct of the previtamin D compound, followed by cycloreversion of the modified adduct under basic conditions [Nevinckx et al., Tetrahedron 47, 9419 (1991); Vanmaele et al., Tetrahedron 41, 141 (1985) and 40, 1179 (1991); Vanmaele et al., Tetrahedron Lett. 23, 995 (1982)], one can expect that the desired 1xcex1-hydroxyvitamin can be contaminated with the previtamin 5(10),6,8-triene and 1 P-hydroxy isomer. One of the most useful C-1 hydroxylation methods, of very broad scope and numerous applications, is the experimentally simple procedure elaborated by Paaren et al. [see J Org. Chem. 45, 3253 (1980) and Proc. Natl. Acad. Sci. U.S.A. 75, 2080 (1978)]. This method consists of allylic oxidation of 3,5-cyclovitamin D derivatives, readily obtained from the buffered solvolysis of vitamin D tosylates, with SeO2/ t-BuOOH and subsequent acid-catalyzed cycloreversion to the desired 1xcex1-hydroxy compounds. Taking into account this synthetic path it is reasonable to assume that the final product can be contaminated with 1xcex2-hydroxy epimer, 5,6-trans isomer and the previtamin D form. 1xcex1-hydroxyvitamin D4 is another undesirable contaminant found in 1xcex1-hydroxyvitamin D2 synthesized from vitamin D2 or from ergosterol. 1xcex1-hydroxyvitamin D4 results from C-1 oxidation of vitamin D4, which in turn is derived from contamination of the commercial ergosterol material. Typically, the final product may contain up to about 1.5% by weight 1xcex1-hydroxyvitamin D4. Thus, a purification technique that would eliminate or substantially reduce the amount of 1xcex1-hydroxyvitamin D4 in the final product to less than about 01.-0.2% would be highly desirable.
The vitamin D conjugated triene system is not only heat- and light-sensitive but it is also prone to oxidation, leading to the complex mixture of very polar compounds. Oxidation usually happens when a vitamin D compound has been stored for a prolonged time. Other types of processes that can lead to a partial decomposition of vitamin D compounds consist of the some water-elimination reactions; their driving force is allylic (1xcex1-) and homoallylic (3xcex2-) position of the hydroxy groups. The presence of such above-mentioned oxidation and elimination products can be easily detected by thin-layer chromatography. Thus, for example, using precoated aluminum silica sheets [with UV indicator; from EM Science (Cherry Hill, N.J.)] and solvent system hexane-ethyl acetate (4:6), the spot of 1xcex1-OH-D2 (Rf 0.27) and its elimination products (Rf""s ca. 0.7-0.9) are visible in ultraviolet light. Also, after spraying with sulfuric acid and heating, an additional spot can be visualized (Rf 0), derived from oxidation products.
Usually, all 1xcex1-hydroxylation procedures require at least one chromatographic purification. However, even chromatographically purified 1xcex1-hydroxyvitamin D2, although showing consistent spectroscopic data, suggesting its homogeneity, does not meet the purity criteria required for therapeutic agents that can be orally, parenterally or transdermally administered. Therefore, it was evident that a suitable method of purification of 1xcex1-hydroxyvitamin D2 is required.
Since it is well known that the simplest procedure that can be used for compound purification is a crystallization process, it was decided to investigate purification of 1xcex1-OH-D2 by means of crystallization. The solvent plays a crucial role in the crystallization process, and is typically an individual liquid substance or a suitable mixture of different liquids. For crystallizing 1xcex1-hydroxyvitamin D2, the most appropriate solvent and/or solvent system is characterized by the following factors:
(1) low toxicity;
(2) low boiling point;
(3) significant dependence of solubility properties with regard to temperature (condition necessary for providing satisfactory crystallization yield); and
(4) relatively low cost.
It was found that highly apolar solvents (e.g. hydrocarbons) were not suitable due to the low solubility of 1xcex1-OH-D2 in them. Quite the reverse situation occurred in highly polar solvent media (e.g. alcohols), in which 1xcex1-OH-D2 showed too high solubility. Therefore, it was concluded that for the successful crystallization of 1xcex1-OH-D2, a solvent of medium polarity is required or, alternatively, a solvent mixture consisting of two (or more) solvents differing considerably in polarity. Interestingly, hexane, so frequently used for crystallization purposes with co-solvents like acetone, ethyl acetate or diethyl ether, was found less suitable for crystallization of 1xcex1-OH-D2. Unusually low yields of crystallization were obtained when hexane-containing solvent systems were used. However, it was discovered that replacement of the hexane in such solvent mixtures with petroleum ether increased significantly the yield of crystals. After numerous experiments it was found that an individual solvent, namely ethyl formate, was most useful for the crystallization of 1xcex1-OH-D2. In addition, binary and ternary solvent systems namely: ethyl acetate-petroleum ether and 2-propanol-hexane-petroleum ether, respectively, also performed well. These solvents are all characterized by low toxicity, and they are very easy to remove by evaporation or other well known methods. In all cases the crystallization process occurred easily and efficiently; and the precipitated crystals were sufficiently large to assure their recovery by filtration.